Measuring viral reverse transcriptase activity

ABSTRACT

In clinical settings as well as in a drug-discovery context, the impact of an agent that may affect reverse transcriptase (RT) can be measured and even quantified by bringing a sample, which may contain an RT, into contact with an RNA template, a primer complementary to the RNA template, and appropriate oligonucleotide-specific primers, under conditions such that they react, in the presence of RT, to form a cDNA product in inverse proportion to the effect of the agent. The amount of any resultant cDNA product then can be measured. The approach is readily implemented as a real-time, quantitative kinetic assay for RT activity.

FIELD OF THE INVENTION

The present invention relates generally to the detection of reversetranscriptase activity and, more particularly, to measuring viralreverse transcriptase quantitatively, in real time, in the context ofdrug screening as well as clinical testing.

BACKGROUND OF THE INVENTION

Silver et al., Nucleic Acids Res. 21: 3593 (1993), disclose an approachto detect a retrovirus, in a biological sample, by PCR amplification ofthe cDNA product of viral reverse transcriptase (RT). By this approach,a cDNA product is synthesized only if viral RT is present, and that cDNAthen is detected by a conventional technique for identifying thepresence of a DNA. Techniques for this purpose include, for example,Southern blot hybridization, visualization by ethidium bromide staining,measurement of incorporated radiolabeled nucleotides, and immunoassayingfor a detectable moiety bound to the amplified DNA.

The techniques of product-enhanced reverse transcriptase assay (PERT,a/k/a “Amp-RT”) and polymerase chain-reaction-based reversetranscriptase assay (PBRT) also have been employed to detect retroviralcontamination, given their 10⁶-fold enhanced sensitivity, relative toconventional RT assays. Thus, the FDA Center for Biologics Evaluationand Research (CBER) has recommended PERT assays for screening patientsfor the presence of RT, and additionally for screening viral vaccine andgene-therapy preparations for RT contamination. Also, U.S. Pat. No.5,849,494 describes the use of these methods for assaying the presenceof human immunodeficiency virus (HIV) in a biological sample. On theother hand, PERT and PBRT present significant drawbacks in a clinicalsetting, because these methods are laborious and cannot be effected inreal time.

The application of TaqMan® technology (PE Biosystems), in conjunctionwith a standard PCR-RT protocol for presence of RT, is well-establishedfor the quantitative measurement of reverse transcriptase. See Ringel etal., J. Clin. Endocrinol. & Metabolism 84: 4037 (1999). Detection of RTactivity with the TaqMan® technique can be effected rapidly andrelatively safely. The TaqMan® technique further represents an advanceover previous methodology because of its considerably increasedsensitivity over conventional RT assays, the ability to shortenmeasurement time to within six hours, while eliminating the need fortoxic chemicals such as radioisotopes and ethidium bromide.

Lovatt et al., J. Virological Methods 82: 185 (1999), report usingTaqMan® PCR technology to detect and quantify viral contamination, inunknown samples, in the laboratory setting. Although the TaqMan®technique is employed in the laboratory setting, it apparently has notbeen used to detect HIV reverse transcriptase. Furthermore, althoughreverse transcriptase is known to be inhibited by various drug agents, amethod of monitoring such agents' effectiveness by assaying RT islacking in the clinical setting.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide anapproach, for detecting viral RT, which is readily adapted to clinicaluse.

It is another object of the present invention to provide for thereal-time monitoring of RT amplification products via a methodology thatis particularly suited to testing agents for RT-affecting activity,e.g., in a high-throughput screening environment.

It is a further object of the present invention to assess, via themonitoring of viral RT activity, the efficacy of an anti-HIV or otheranti-viral drug or putative therapeutic agent.

It is similarly an object of the invention to provide a diagnostictechnique which can be performed in a clinical setting, rapidly andsafely, to identify a range of pathogens of common medical concern.

It is still another object of the present invention to detect thepresence of RT that is resistant, or to measure the level of resistance,to a known RT inhibitor.

In accomplishing these and other objectives, there has been provided, inaccordance with one aspect of the invention, a method for measuring thepresence of RT in a sample. The sample is tested for the presence of RTby contacting the sample with (i) an RNA template, such aspolyadenylated RNA, (ii) a primer that is complementary to the RNAtemplate, for example, oligo-dT, and (iii) a plurality ofoligonucleotide-specific primers, at about a temperature preferably lessthan the deactivation temperature of the RT, which is typically in therange of about 37° C. for HIV RT. If RT is present in the sample, thencDNA is produced and can be detected accordingly.

In another embodiment of the present invention, a method is provided formeasuring the effect of a test agent on the activity of an RT in asample. The sample, which may contain an RT, is brought into contactwith an RNA template, preferably human, a complementary primer, andplurality of oligonucleotide-specific primers such that cDNA is formedonly if RT is present in the sample in an amount inversely proportionalto the effect of said test agent. The sample is preferably taken from ahuman patient and is tested in the presence of HIV RT in real time in aclinical setting.

In this vein, one skilled in the art will understand that RT in asample, preferably from a patient, can be tested to determine whetherthe RT in the sample has developed resistance to a known RT inhibitingtest agent. This can be accomplished by measuring the effect of a knownRT inhibiting test agent on the activity of RT in a sample, comparingthat activity against the activity of RT in the absence of the known RTinhibitor and/or the activity of the RT in the presence of an RTinhibitor which is known to completely inhibit the RT activity of thesample.

Other objects and advantages of the invention will become apparent byreview of the detailed description of preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cDNA calibration curve.

FIG. 2 depicts the inhibition of rHIV RT by increasing concentrations oftest agent.

FIG. 3 similarly depicts the inhibition of rHIV RT by increasingconcentrations of test agent.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In a preferred embodiment of the present invention, determining thepresence of RT in a sample, such as a biological sample from a patient,entails introducing the sample to a reaction mixture that contains totalhuman heart poly adenylated RNA, oligo-dT primer and dNTPs. If RT ispresent in the sample, then it will transcribe cDNA that corresponds tothe total human heart poly A-RNA. Conversely, no cDNA product will beformed if RT is absent from the sample. Formation of any cDNA will beproportional to the concentration of any active RT present in thesample.

In accordance with the present invention, formation of cDNA, by virtueof the presence of RT in the sample, is followed by amplification anddetection of the cDNA, preferably by means of a Taqman® technique wellknown in the art. It also is possible to detect the amplified cDNAproduct by other techniques well known in the art, including, but notlimited too, Southern blot hybridization, ethidium bromide staining,measurement of incorporated radiolabeled nucleotides, immunoassaying fora detectable moiety bound to the amplified DNA, and measurement offluorescence.

The present invention is applicable to measuring RT enzyme for a broadrange of pathogens. Thus, the method can be employed in relation to allRNA viruses including, without limitation, retroviruses, flaviviruses,and yellow fever viruses. Illustrative flaviviruses in this regard arethe West Nile virus and the encephalitis viruses, such as those strainsassociated with St Louis encephalitis, western equine encephalitis,Japanese equine encephalitis, and tick borne encephalitis.

Because the Taqman® assay is sensitive down to 10 copies of DNA, it isseveral log fold more sensitive than conventional PCR protocols,including gel based separations of the PCR amplicon. Further, theTaqman® assay is amenable to rapid throughput, and allows fordetermination of both RT activity and inhibition of RT activity, asdescribed below.

The underlying basis for the Taqman® assay is that taq polymerasepossesses both endonuclease and polymerase activities. Once the cDNA isformed by the action of RT, oligonucleotide-specific primers are usedfor the amplification aspect of PCR. Hybridizing “between” the primerson the cDNA, the Taqman® probe contains a reporter fluor, which can beFAM, at the 5′ end of the oligonucleotide, and a quencher dye, which canbe TAMRA, at the 3′ end of the probe. The probe itself is notfluorescent, due to intramolecular fluorescence quenching. Thus, whenthe probe is intact, the proximity of the reporter fluor to the quencherdye results in suppression of the reporter fluorescence, primarily byForster-type energy transfer. As the PCR proceeds, amplification of thetarget cDNA occurs and the DNA polymerase, during extension of theprimer sequences, effects the 5′ cleavage of the probe reporter,resulting in a reversal of quenching of fluorescence of the reporterand, hence, in a measurable fluorescent signal of cDNA production.

The cycle number at which the fluorescence emission of the PCR reactionexceeds the background fluorescence level is referred to as thethreshold cycle (C_(T)) value and is calculated in real time. The C_(T)value is dependent upon both the amount of PCR template and cDNA presentin the reaction mixture. Thus, the RT activity in the sample can bequantified by comparing the C_(T) value of an unknown, measured sampleagainst the C_(T) value of sample, run under the same conditions, havinga known amount of cDNA.

In a preferred embodiment, the Taqman® protocol is used to detect thepresence of recombinant HIV (rHIV) RT. In this regard, oligo dT is usedas a template for rHIV RT. Oligo dT is complementary to thepolyadenylated RNA-component of the total human heart RNA which presentin the reaction mixture. rHIV RT forms cDNA from the oligo dT/RNAcomplex in the presence of dNTP nucleotides. If a patient sample is tobe tested for the presence of HIV RT, an extract prepared from thepatient sample is added, in place of rHIV-RT.

Some RTs tend to be heat-sensitive, and so the reaction described abovemust be performed under cycling conditions which do not destroy theactivity of the RT, which occurs at a temperature of about 37° C. forrHIV RT, for example. Thus, the temperature cycling conditions for rHIVRT were about 25° C. for 10 minutes, followed by about 37° C. for 60minutes, and finally 95° C. for 5 minutes. At 95° C., the activity ofrHIV RT was destroyed. Note that the standard Taqman® temperaturecycling conditions will also destroy the activity of rHIV RT. Typically,this aspect of the protocol is effected outside of the Taqman®instrument on a programmable PCR thermocycler.

Next, an aliquot of the cDNA formed in the RT step above is brought intothe Taqman® instrument (PE Model 7700) and put into a reaction mixturewhich contains Taqman® Universal master mix (taq polymerase in buffer),beta actin-specific forward primer, beta actin-specific reverse primer,and beta actin Taqman® probe. The PCR step takes place in the instrumentunder standard Taqman® PCR conditions. Although, as described above, thepresent method is depicted as a two test tube reaction, one skilled inthe art will realize that these steps can be combined in a singlereaction vessel.

In another embodiment of the present invention, efficacy is ascertainedfor a test agent that can inhibit the activity of RT. In this context,the test agent preferably is added when the template RNA, the primerwhich is complementary to the template RNA, and the dNTPs are mixed inthe presence of the sample. Accordingly, although the sample may containactive RT, cDNA transcription by the RT can be inhibited by the presenceof the test agent in the reaction mixture.

The inhibiting properties of the test agent are reflected in an increasein C_(T) value for the sample which contains the test agent. The morepotent the inhibition of RT activity by the test agent, the higher theC_(T) value from the Taqman® assay, measured against a control reactioncontaining no test agent. Because RT inhibition can be measured in realtime, the present invention is optimally suited for real time drugscreening of test agents which may have potential RT inhibitingproperties. Additionally, the present method can be performed utilizinga biological sample from a patient with different known RT inhibitingagents, e.g., nucleoside and nucleotide analogs and non-nucleosidereverse transcriptase inhibitors, or combinations thereof, to screen andmeasure the patient RT for resistance against these RT inhibiting drugsin real time, thus leading to more informed drug dosage anddispensation.

In a clinical setting, in situ measurement of the efficacy of testagents allows for higher throughput of samples and subsequent efficiencyin targeting promising test agents which exhibit inhibiting activity.Thus, purification of inhibiting agents can be tracked in addition todetermining and quantitating the specific activity of the inhibitingagents. Similarly, drug choices for patients who may have built upresistance to specific drugs can be assayed without resulting in a lossof treatment time that can result from drug experimentation performed todetermine if the patient evinces resistance. Toxicity and side effectsof drugs on patients can further be limited as dosage testing ofinhibiting agents can be performed to determine the optimal dosage ofthe inhibiting drug.

Additionally, the present invention can be performed, ex vivo, in aclinical setting on human patients. Because the present invention can beperformed ex vivo, the activity of RT and efficacy of test agents isdetermined in a way that is non-toxic to the patient. As noted before,because the present invention measures RT activity or inhibition thereofin real time, assaying patient samples for the presence of specificpathogens can be performed in an extremely prompt manner, typically lessthan one hour.

The following, non-limiting example further illustrates advantages ofthe present invention. These solutions were employed in the protocol:

1. TaqMan® RT Reagent kit, which contains 10× reaction buffer, magnesiumchloride stock solution, and RNAse inhibitor—Perkin Elmer catalogue No.N808-0234;

2. rHIV RT—Worthington Biochemical catalogue No. 5006 (500 U at 12.5U/μL);

3. Total Human Heart RNA—Ambion catalogue No. 7966 (100 μg at 1 mg/ml);

4. 100 mM dNTP Mix (25 mM each NTP)—Perkin Elmer catalogue No.N808-0261;

5. Oligo dT (17-mer);

6. Random Hexamer (oligo (dNTP)6);

7. 2× TaqMan® Universal Master Mix—Perkin Elmer catalogue No. PE4304437;

8. TaqMan® Beta Actin Reagents, which includes specific forward andreverse oligonucleotide primers for beta actin, plus the TaqMan® probefor beta actin—Perkin Elmer catalogue No. 401846; and

9. Beta Actin Plasmid DNA (American Type Tissue Culture catalogue No.769559R).

A solution of 10 mM dNTP was prepared by diluting 1:10 dilution of 100mM dNTP solution using water as the diluent. A 50 μM stock solution ofoligo dT in water also was prepared.

An rHIV RT solution (Master Mix solution)was prepared which contained 5μL 10× RT reaction buffer, 11 μL 25 mM MgCl₂, 10 μL 10 mM dNTP workingstock solution, 2.5 μL 50 μM oligo dT, 1 μL RNAse inhibitor and 1.25 μLrHIV RT. The total volume of the Master Mix solution was 30.75 μL.Alternatively, random hexamer may be added in place of oligo dT. OligodT is the preferred primer, however.

Water plus test agent (totaling 18.25 μL) plus 1 μL total human heartRNA, for example:

1 μL test agent+17.25 μL water+1 μL total human heart RNA;

2 μL test agent+16.25 μL water+1 μL total human heart RNA;

5 μL test agent+13.25 μL water+1 total human heart μL RNA;

10 μL test agent+8.25 μL water+1 total human heart μL RNA;

15 μL test agent+3.25 μL water+1 total human heart μL RNA,

were added to 30.75 μL of the Master Mix solution. Thus, the reactionmixture contained 50 μL total solution volume.

The controls for the present example included a positive controlcontaining 18.25 μL water plus 1 μL RNA, and a negative controlcontaining 19.25 μL water.

The transcriptase reaction was initiated by placing the reaction mixturein a MWG Thermocycler, or equivalent, under thermocycling conditions at25° C. for 10 minutes, then at 37° C. for 60 minutes, and finally at 95°C. for 5 minutes. This final thermocycling step deactivated the rHIV RT.Next 5 μL of the post-thermocycle RT reaction mixture, which presumablycontained cDNA, were added to 45 μL of the Master Mix solution. TheMaster Mix Solution contained 25 μL 2×Taqman Universal Master Mix, 5 μLbeta Actin Forward Primer, 5 μL Beta Actin Reverse Primer and 5 μLwater. Thus, the total PCR reaction mixture contained 50 μL.

The PCR reaction was carried out in the Perkin Elmer 7700 TaqManinstrument, as recommended by the manufacturer, under the followingconditions: at 50° C. for 2 minutes; then at 95° C. for 10 minutes. 40cycles were then performed at 95° C. for 15 seconds followed by 60° C.for 1 minute. Upon completion, the data were plotted and visualized asdescribed in the Perkin Elmer 7700 TaqMan instrument manual.

Increase in fluorescence was monitored as a function of cycle number.The data was presented as relative fluorescence versus C_(T). Acorrelation between C_(T) value and number of copies of DNA wasdetermined from a calibration curve prepared using a known amount ofDNA. For this determination, beta Actin DNA was used in place of the RTreaction mixture in the PCR reaction step. The useful range of betaactin DNA with which to prepare the calibration curve was 5 ag(atograms) which corresponds to approximately 1 copy of DNA to 500 fg(femtograms) which corresponds to 9.5×10⁴ copies of DNA.

The number of copies of DNA was based on the following observation: Onemicrogram (1 μg) of PBR322 plasmid DNA is equivalent to 36 pmol of PBR322 plasmid DNA (2.2×10¹¹ copies of DNA). PBR322 plasmid DNA encompasses4360 bp. Beta actin plasmid DNA encompasses 5000 bp.

The TaqMan® calibration curve is shown in FIG. 1. In the figure, knownamounts (known copy numbers) of beta-actin DNA were used for the PCRcomponent of the assay protocol and the observed threshold value (C_(T))recorded. Note that with decreasing copy number, the C_(T) value becomesprolonged. The calibration curve assay is linear down to at least 1 copyof DNA and extends to at least 100,000 copies of DNA. The curve was fitby apparent linear regression (this is a linear/log plot) and shows aregression fit of 0.99. The equation of the line is Y=−3.12(X)+36, whereX is the DNA copy number and 36 is the C_(T) value at infinitely low(less than one) copy of DNA.

In the EXAMPLES below, the above protocol was used, with minormodifications which will be obvious to one skilled in the art.

EXAMPLE I

Example I illustrates the increase in fluorescence as a function ofcycle number using a test agent in increasing final concentrations of 0,1%, 5%, 10% (v/v). The C_(T) value of the positive control is indicatedby a horizontal black line. This C_(T) value is the midpoint of theincrease in fluorescence above background. As illustrated in FIG. 2, thepositive control sample showed a C_(T) value of 24 cycles. The remainingcurves of FIG. 2 demonstrate inhibition of rHIV RT in the presence of atest agent proprietary to applicant. At higher concentrations of testagent, the C_(T) value became increasingly larger, indicating that therHIV RT activity was being progressively inhibited.

EXAMPLE II

Another example of rHIV RT inhibition in the presence of a secondanti-viral test agent proprietary to applicant. As shown in FIG. 3,samples that crossed the threshold at C_(T) 26 are the RT PCR control(no test agent) and 1% (v/v) test agent (as final concentration in theRT reaction mixture). The 5% (v/v) test agent sample crossed thethreshold at cycle 33 and the 10% (v/v) test agent never crossedthreshold, indicating 100% inhibition of rHIV RT activity.

Inhibition caused by the test agent can be expressed in terms of thenumber of copies of DNA present in the PCR reaction mixture. Thus:

Sample CT # Copies DNA % Inhibition Control 26 1701 0 1% test agent 261701 0 5% test agent 33  10 99.4 10% test agent >40   0 100

EXAMPLE III

The following example demonstrates the inhibition efficacy of AZT on asample containing rHIV RT

AZT (a nucleotide analog of dT) in the range of 250 μM to 1.25 mM wasadded to the rHIV-RT reaction mixture in place of dT. When the AZT wasadded, no amplification of cDNA was observed after the PCR step.Similarly, when AZT was included in the PCR reaction mixture in place ofdT, NO amplification of the cDNA was seen after the PCR step.

Finally, decreasing concentrations of AZT were added to the RT reactionmixture in the presence of a fixed concentration of dT. The final AZTconcentration ranged from 1.25 mM down to 250 μM. At all concentrationsof AZT, the observed C_(T) was greater than 40 cycles, indicating 100%inhibition of activity.

While preferred embodiments have been illustrated and described, itshould be understood that changes and modifications can be made thereinin accordance with one of ordinary skill in the art without departingfrom the invention in its broader aspects as defined in the followingclaims.

What is claimed is:
 1. A method for measuring the effect on reversetranscriptase of an agent affecting a sample that may contain a reversetranscriptase, comprising (a) bringing said sample into contact withtemplate RNA, a primer complementary to said template RNA, and aplurality of oligonucleotide-specific primers under conditions such thatthey react, in the presence of a reverse transcriptase, to form a cDNAproduct in inverse proportion to the effect of said agent, and (b)measuring the amount of said cDNA product.
 2. The method of claim 1,wherein the template RNA is a polyadenylated RNA and the primercomplementary to said template RNA is an oligo-dT primer.
 3. The methodof claim 1, wherein step (b) comprises amplifying said cDNA product andthen measuring said amount in relation to cDNA product obtained in theabsence of said agent.
 4. The method of claim 2, wherein saidpolyadenylated RNA is human RNA.
 5. The method of claim 2, wherein saidconditions include a reaction temperature of 37° C. or less.
 6. Themethod of claim 3, wherein said reverse transcriptase is an HIV reversetranscriptase.
 7. The method of claim 3, wherein said reversetranscriptase is recombinant HIV reverse transcriptase.
 8. The method ofclaim 3, wherein said sample is comprised of biological materialinfected by HIV.
 9. The method of claim 2 wherein step (a) is precededby obtaining said sample from a patient.
 10. The method of claim 3wherein step (a) is preceded by obtaining said sample from a patient.11. A method for measuring reverse transcriptase in a sample that maycontain an HIV reverse transcriptase, comprising (a) bringing saidsample into contact with template RNA, a primer complementary to saidtemplate RNA, and a plurality of oligonucleotide-specific primers underconditions such that they react, in the presence of a reversetranscriptase, to form a cDNA product, wherein said conditions include areaction temperature less than about the deactivation temperature ofsaid reverse transcriptase, and (b) detecting the presence of cDNAproduct.
 12. The method of claim 10, wherein the template RNA is apolyadenylated RNA and the primer complementary to said template RNA isan oligo-dT primer.
 13. The method of claim 11 wherein the reactiontemperature is less than about 37° C.
 14. The method of claim 10,wherein step (b) comprises amplifying and then measuring said cDNAproduct.